magnetic cross-linked enzyme aggregates (cleas) of candida antarctica lipase. an efficient and...

RESEARCH ARTICLE

Magnetic Cross-Linked Enzyme Aggregates(mCLEAs) of Candida antarctica Lipase: AnEfficient and Stable Biocatalyst forBiodiesel SynthesisAlvaro Cruz-Izquierdo1a, Enrique A. Pico1, Carmen Lopez1,2*b, Juan L. Serra1,Mara J. Llama1

1. Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao,Spain, 2. IKERBASQUE, Basque Foundation for Science, Bilbao, Spain

*[email protected]

a Current address: Department of Chemistry, University of Bath, Bath, United Kingdomb Current address: Department of Chemistry and Food Technology, Universidad Politecnica de Madrid,Madrid, Spain

Abstract

Enzyme-catalyzed production of biodiesel is the object of extensive research due to

the global shortage of fossil fuels and increased environmental concerns. Herein

we report the preparation and main characteristics of a novel biocatalyst consisting

of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B

(CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for

the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this

purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles

of magnetite which the surface was functionalized with NH2 groups. The resulting

biocatalyst combines the relevant catalytic properties of CLEAs (as great stability

and feasibility for their reutilization) and the magnetic character, and thus the final

product (mCLEAs) are superparamagnetic particles of a robust catalyst which is

more stable than the free enzyme, easily recoverable from the reaction medium and

reusable for new catalytic cycles. We have studied the main properties of this

biocatalyst and we have assessed its utility to catalyze transesterification reactions

to obtain biodiesel from non-edible vegetable oils including unrefined soybean,

jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil)

conversions near 80% were routinely obtained at 30C after 24 h of reaction, thisvalue rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily

recovered from the reaction mixture and reused for at least ten consecutive cycles

of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs

OPEN ACCESS

Citation: Cruz-Izquierdo A, Pico EA, Lopez C,Serra JL, Llama MJ (2014) Magnetic Cross-LinkedEnzyme Aggregates (mCLEAs) of Candida ant-arctica Lipase: An Efficient and Stable Biocatalystfor Biodiesel Synthesis. PLoS ONE 9(12):e115202. doi:10.1371/journal.pone.0115202

Editor: Pratul K. Agarwal, Oak Ridge NationalLaboratory, United States of America

Received: July 1, 2014

Accepted: November 19, 2014

Published: December 31, 2014

Copyright: 2014 Cruz-Izquierdo et al. This isan open-access article distributed under the termsof the Creative Commons Attribution License,which permits unrestricted use, distribution, andreproduction in any medium, provided the originalauthor and source are credited.

Data Availability: The authors confirm that all dataunderlying the findings are fully available withoutrestriction. All relevant data are within the paperand its Supporting Information files.

Funding: This work was supported by grants fromthe University of the Basque Country (UPV/EHU,project GIU11/25 and scholarship for AC-I, http://www.ehu.es), the Spanish Ministry of Economyand Competitiveness (project CTQ2011-25052,http://www.mineco.es), the Basque Government(project SAIOTEK S-PE12UN041, http://www.euskadi.net), Ikerbasque, the Basque Foundationfor Science (fellowship for CL, www.ikerbasque.net) and European Union (project Energreen,POCTEFA EFA217/11, http://www.poctefa.eu/).The funders had no role in study design, datacollection and analysis, decision to publish, orpreparation of the manuscript.

Competing Interests: The authors have declaredthat no competing interests exist.

PLOS ONE | DOI:10.1371/journal.pone.0115202 December 31, 2014 1 / 22

prepared from CALB can become a powerful biocatalyst for application at industrial

scale with better performance than those currently available.

Introduction

Due to the global shortage of fossil fuels, a consequent excessive rise in the price of

crude oil and increased environmental concerns, a rapid growth in biodiesel

production has being observed [1]. Biodiesel is a clean-burning diesel fuel

composed of a mixture of alkyl esters of long-chain fatty acids which is typically

produced from nontoxic, renewable biological resources such as vegetable oils,

animal fats, or even used cooking oils [2]. Nowadays, the alkali-catalyzed

transesterification of triglycerides present in oils or fats is the most common way

to produce biodiesel at industrial scale due to its high conversion and kinetics.

However, when these raw materials contain an elevated percentage of water or free

fatty acids, undesirable reactions drastically reduce the yield and quality of the

resulting biodiesel [2]. Thus, enzyme-catalyzed production of biodiesel by lipases

is more advantageous over the chemical counterpart since it is chemically

selective, reaction is carried out at lower temperature and under milder conditions

and downstream processes are more environmentally friendly [3].

Lipases constitute a group of versatile enzymes that catalyze the hydrolysis of

lipids in aqueous media, but in organic media they can catalyze synthetic

reactions, including interesterifications between triglycerides and alcohols to

produce glycerin and alkyl-esters of long chain fatty acids, i.e., biodiesel. However,

free lipases are easily inactivated in organic solvents and difficult to recover for

reuse [4]. Candida antarctica lipase B (CALB) is an extracellular monomeric

globular (33.5 kDa) enzyme which is not as efficient as other lipases in

hydrolyzing triglycerides, but it is highly stereospecific towards both ester

hydrolysis and synthesis. Due to its efficiency and high selectivity this lipase has

been immobilized by several methods (especially by covalent attachment to silica

gel, celite or activated nanoparticles) and it is being used in a wide range of

applications replacing industrial synthetic processes [5].

Many strategies have been recently applied to immobilize CALB. One of the

most successful consists of covalent linking of the enzyme to the surface of iron

oxide magnetic nanoparticles (MNPs) [6, 7]. The high specific surface of MNPs

favors the binding efficiency of the enzyme, and the superparamagnetic behavior

of the support permits the easy and selective recovery of the biocatalyst using a

magnet and its subsequent reuse in more catalytic cycles [8, 9]. On the other hand,

the support-free methodology consisting of the formation of cross-linking enzyme

aggregates (CLEAs) of lipase with glutaraldehyde [10, 11] is a simple technique

which does not require the use of highly pure enzymes. It has a broad scope and

affords robust, recyclable catalysts that exhibit high activity retention, enhanced

thermal stability, better tolerance to organic solvents, and enhanced resistance to

Magnetic CLEAs of Lipase for Biodiesel Synthesis


autoproteolysis due to the rigidification of the tertiary structure of the enzyme

[1215]. Due to the large size of the aggregates, CLEAs can be recovered from the

reaction medium by centrifugation or filtration, although this process leads to the

increase of CLEAs size and formation of clusters (clumping) resulting in internal

mass-transfer limitations [16, 17].

In recent years, lipases immobilized on MNPs or in CLEAs have been

successfully used to obtain biodiesel. Xie and Ma [18, 19] reported 90%

conversion to biodiesel using covalently immobilized Thermomyces lanuginosus

lipase on amino-functionalized MNPs. Also Burkholderia cepacia lipase immo-

bilized to MNPs of Fe3O4 yielded complete conversion to biodiesel [20]. In

addition biodiesel was obtained from olive oil using CLEAs of crude preparations

from Photobacterium lipolyticum lipase M37 [21]. Lai et al. [22] also reported

85.7% conversion to biodiesel from microalgal oil using an ionic liquid (1-butyl-

3-methylimidazolium hexafluorophosphate) as a solvent and CLEAs obtained

from crude preparations of Penicillium expansum.

Recently, our group has developed a method for the preparation of magnetic

cross-linked enzyme aggregates (mCLEAs) of CALB. This method consists of the

cross-linking of insolubilized lipase to aminated MNPs by glutaraldehyde [23].

The resulting mCLEAs fulfill the main benefits of magnetic biocatalysts and

CLEAs, as they show improved thermal and storage stabilities, and can be reused

after their recovery from the reaction mixture with a magnet. Also mCLEAs of a-

amylase from Bacillus sp. [16] and lipase from Aspergillus niger [24] were

successfully prepared and used to hydrolyze starch and to obtain glycerol

carbonate, respectively.

In this paper, the main magnetic properties of mCLEAs were assessed as well as

the utility of this robust biocatalyst to obtain biodiesel from several non-edible

vegetable oils. Finally, the enhanced stability of our mCLEAs of CALB was

ascertained by reusing the magnetic enzyme to catalyze the production of

biodiesel in consecutive 24 h batch reactions. A similar procedure to that detailed

here to prepare mCLEAs of CALB can be easily expanded to prepare mCLEAs of

other enzymes of interest.

Materials and Methods

MaterialsCALB L, the lipase B of Candida antarctica (CALB, EC 3.1.1.3, 19.1 U/mg protein;

7.50 mg protein/ml) was kindly provided by Novozymes (Bagsvaerd, Denmark).

HyperLadder I markers were purchased from Bioline (Randolph, MA, USA).

Tween 20 and Tween 80 were obtained from Panreac (Barcelona, Spain). 3-

Aminopropyltriethoxysilane (APTS), NaBH4, FeCl2, FeCl3, dimethylsulfoxide

(DMSO), sodium dodecyl sulphate (SDS), p-nitrophenyl acetate (pNPA), bovine

serum albumin, and Triton X-100 were purchased form Sigma-Aldrich (St. Luis,

MO, USA). Coomassie Blue was obtained from GE Healthcare (Uppsala, Sweden).

All other chemicals were supplied by Merck (Darmstadt, Germany). Non-edible



oils (unrefined soybean, jatropha and cameline) were obtained from Bunge

Iberica, S.A. (Zierbena, Spain), Jatropha Hispania, S.L. (Toledo, Spain) and

Camelina Company (Madrid, Spain), respectively. Olive oil used as a control was

purchased from Carbonell (Madrid, Spain) and waste frying oil was obtained

from local restaurants.

Synthesis and functionalization of magnetic nanoparticles (MNPs)Magnetic nanoparticles of magnetite (Fe3O4) were synthesized by coprecipitation

of iron salts in alkaline medium [25]. An aqueous solution, 50 ml, containing

0.36 M FeCl2 and 0.72 M FeCl3 was pumped at 5.0 ml/min (LKB Pump P-1,

Pharmacia, Uppsala, Sweden) into 450 ml of 1 M NH4OH solution under

continuous mechanic stirring at room temperature (S1 Video). The obtained

black precipitate was separated from the liquid phase using a magnetic field, and

then magnetically washed thrice with ultrapure water (MilliQ, Millipore Co.,

Bedford, MA, USA) and twice with phosphate buffered saline (PBS, 100 mM

sodium phosphate, 150 mM NaCl, pH 7.4). All solutions used for the synthesis of

MNPs were prepared using MilliQ water. Moreover, a N2 gas stream was

continuously bubbled through all solutions during the process. The resulting

magnetic nanoparticles contained OH groups on their surface [26, 27]. In order

to functionalize them with NH2 groups, MNPs (300 mg, dry weight) were

incubated with 30 ml of 2% (v/v) APTS at 70 C with orbital shaking (200 rpm).

After 24 h, they were washed thrice with PBS and maintained in the same buffer at

4 C until use.

Characterization of MNPsPhase identification was done by X-ray diffraction studies (XRD, Philips

diffractometer PW1710, Almelo, The Netherlands). The crystallite size was

calculated from the X-ray line broadening using Scherrers and Warrens

equation:

D~K:l

b: cos h

where K takes the value of 0.89 considering spherical particles; l is the wavelengthof X radiation used (0.54 nm); h is the Bragg angle; b is the net diffraction peakbroadening in radians being determined from:

b2~b2obs{b2ins (Warren)

b~bobs{bins (Scherrer)

where bobs is the observed broadening (experimental) and bins the instrumentalbroadening.

The particle size and morphology were determined by transmission electron

microscopy (TEM, JEOL 1010, Peabody, MA, USA). The density of NH2 groups



on the nanoparticles surface was determined by a colorimetric assay using p-

nitrobenzaldehyde [28]. The magnetic characteristics were measured using

different devices: for the calculation of magnetic saturation (Ms) 2 K hysteresis

loops were performed using a vibrating sample magnetometer (VSM) in a

superconducting magnet (14 T) cooled by a closed circuit of He (CFMS,

Cryogenic Ltd., London, UK). To calculate coercivity (Hc) and remanence (Mr) an

electromagnet was used at room temperature and moderate fields (0.9 T). The

average size of magnetic particles was calculated from the magnetization data

according to Langevins equations adjusted for non-interacting superparamag-

netic model. MNPs dry weight and concentration were analyzed using a vacuum

concentrator (Savant SpeedVac concentrator, Thermo Scientific, Waltham, MA,

USA).

Preparation of magnetic CLEAs from CALBIn order to obtain magnetic CLEAs, 10 mg (dry weight) of MNPNH2 were

incubated with different concentrations of CALB (from 0.025 to 1.2 mg protein/

mg MNPNH2) in the presence of precipitant agents and detergents in a final

volume of 15 ml. The precipitant agents used were 80% (v/v) ethanol, 80% (v/v)

2-propanol, 80% (v/v) acetone in water at 4 C, or ammonium sulphate at

concentration in the range of 12.5 to 125 mmol/mg protein at 25 C. The detergent

concentrations used were 05 mM Triton X-100, 00.1 mM Tween 20, 00.1 mM

Tween 80 or 025 mM SDS. In all cases, the samples were mixed continuously at

30 rpm using an Intelli-Mixer RM-2, Elmi Ltd. (Riga, Latvia). After 5 min, a

2.5 M glutaraldehyde solution was added (final concentration from 0 to 500 mM)

and the suspension was maintained in agitation for 1 to 24 h. After this time, the

mCLEAs were removed from the liquid phase using magnetic field, and mCLEAs

were washed thrice with PBS. Both the Schiffs bases and the unreacted aldehyde

groups were reduced for 2 h at room temperature with rotational mixing with

NaBH4 (0.75 mg/mg MNPNH2) dissolved in 100 mM carbonate/bicarbonate

buffer, pH 10.0. In order to minimize non-specific interactions mCLEAs were

then magnetically washed for 10 min firstly with 2 M NaCl in PBS and then with

1% (v/v) Triton X-100 in PBS as reported by Mateo et al. (2000) [29]. After each

step, the excess of reagents and possible unretained enzyme were removed by

washing thrice with PBS for 10 min. At the end of the immobilization process, the

final concentration was 2 mg mCLEAs/ml. The magnetic catalyst was stored at

4 C in PBS for future use.

Magnetic biocatalysts at different formation steps were incubated at 100 C for

5 min, and then, the liquid phase was collected and analyzed by sodium dodecyl

sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [30] using homoge-

neous 8% acrylamide gels revealed with silver (Bio-Rad, Hercules, PA, USA). The

amount of immobilized lipase was calculated by measuring the protein remaining

in solution at 595 nm (Bio-Rad protein Assay kit, Hercules, PA, USA).



Enzyme assaysEsterase activity with pNPA

The activity of soluble and immobilized CALB was assayed with p-nitrophenyl

acetate (pNPA) as a substrate according to the protocol reported by Gao et al. [31]

with minor modifications. Specifically, soluble or immobilized lipase (12 mg

protein) was added to a reaction mixture which contained 10 ml of pNPA

(100 mM in DMSO) in 980 ml of PBS. The reaction mixture was maintained for

15 min at room temperature with rotational mixing at 30 rpm (Intelli-Mixer RM-

2, Elmi Ltd., Riga, Latvia). Samples were withdrawn every 5 min (in the case of

immobilized lipase the magnetic biocatalysts were separated from the liquid phase

using a magnet) and the appearance of p-nitrophenol (pNP) was measured

spectrophotometrically (Beckman Coulter DU 800, Brea, CA, USA) at 405 nm

(epNP59.43 mM21?cm21). One unit (U) of esterase activity was defined as the

amount of enzyme that catalyzes the appearance of 1 mmol pNP per min under

the assay conditions.

Transesterification of olive oil

Olive oil (0.2 g) and 2-propanol (1:6 molar ratio oil:alcohol) were incubated with

1% (w/w of oil) of magnetic catalyst. The reaction was maintained at 40 C and

30 rpm with rotational mixing. The initial reaction rate for biodiesel production

was assessed by withdrawing aliquots (5 ml) of the liquid phase at defined intervals

which were analyzed by high-performance liquid chromatography (HPLC) as

indicated below. Transesterification activity was defined as mg of oil transformed

to biodiesel (fatty acid propyl esters, FAPEs) per mg of MNP-NH2 and time (h)

considering the initial reaction rate of transesterification.

Synthesis of biodiesel

FAPEs were produced from vegetable oil (0.2 g) using 2-propanol as alkyl donor

(1:6 molar ratio oil:alcohol) and 1% (w/w of oil) of magnetic catalyst in a solvent-

free reaction. The reaction was followed by withdrawing aliquots (10 ml) of the

liquid phase. Semi-quantitative analysis of samples was performed by thin layer

chromatography (TLC) and the quantitative analysis using control olive oil was

performed by HPLC as described below.

Analytical methodsHPLC analysis

Five ml of samples withdrawn from the reaction mixture were diluted in 250 ml of

n-hexane and analyzed by HPLC (Waters Corporation, Milford, MA, USA) using

a diode array detector according to Holcapek et al. [32]. The C18 column (5 mm,

4.66250 mm, Tracer Lichrosorb RP18) was eluted with 100% pure methanol(phase A) and a mixture of propanol:n-hexane (5:4 v/v) (phase B) at a flow rate of

0.5 ml/min using a gradient from 0 to 50% phase B in 25 min. Peaks were

detected at 205 nm.



Thin layer chromatography (TLC) analysis

A semi-quantitative analysis of non-edible or waste frying olive oil was also

assessed using TLC. Silica gel 60 coated plates (Merck, Darmstadt, Germany) were

activated for 30 min at 100 C and 0.5 ml samples were applied. The ternary

mixture n-hexane:ethyl acetate:acetic acid (90:10:1 v/v/v) was used as the eluting

phase [33]. After chromatography development (about 40 min), plates were air

dried at room temperature, and then immersed for 1 min with gentle orbital

shaking in a solution of 0.02% (w/v) Coomassie Blue R-350 [34], prepared in

acetic acid:methanol:H2O (1:3:6 v/v/v) as indicated by the manufacturer. Finally,

the plates were air dried at room temperature. Spots corresponding to substrates

and products of the transesterification reaction were identified by using

appropriate reference external standards (triolein, 1,3-diolein, 1-oleyl-rac-

glycerol, oleic acid and propyl oleate) run in parallel.

Results and Discussion

Magnetic nanoparticles characterizationMNPs size was analyzed by XRD and TEM. X-ray diffraction patterns showed that

magnetite constitutes the only crystalline phase present both in MNPOH and

MNPNH2 (Fig. 1), with a crystallite size of 11.2 nm and 11.1 nm, respectively,

according to Scherrers equation, and 9.5 nm and 9.4 nm, respectively, according

to Warrens equation. These results indicated that functionalization of MNPs with

NH2 groups did not change the phase, nor the crystallite size of the magnetic

support. The shape and size of the MNPs was confirmed by TEM (Fig. 2), which

showed that both types of MNPs are spherical particles with a mean diameter

around 10 nm, indicating that coating their surfaces with NH2 groups did not

result in observable differences in their size, shape or aggregation state.

The surface areas were theoretically calculated from the particle size

distribution provided by TEM data (Table 1). The results were similar to those

reported by other authors, as Harris et al. [35] who determined a surface area of

110 m2/g for 8.7 nm nanoparticles, or Sahoo et al. [36] who calculated 93 m2/g

for particles with mean diameter of 1012 nm.

The density of NH2 groups on the surface of particles was determined using

naked MNPs as a control (Table 1). The calculated density of NH2 groups on

functionalized nanoparticles (43 mmol/g) was very similar to that reported by del

Campo et al. [28] who found approximately 45 mmol/g of MNPs. These authors

also calculated the density of NH2 groups by elemental analysis obtaining higher

values. This result may suggest that some NH2 groups do not react with 4-

nitrobenzaldehyde, and therefore all the amine groups are not available for the

interaction with the enzyme. In this case, the calculated density would represent

the functionally available groups for the enzyme immobilization.

Magnetic behavior of MNPs was analyzed at different magnetic fields and two

temperatures: one below the blocking temperature of magnetite nanoparticles

(i.e., the temperature below which magnetite nanoparticles show ferromagnetic



Fig. 1. XRD spectrum of magnetic nanoparticles. MNPs were coated with OH (solid line), and NH2(dotted line) groups.

doi:10.1371/journal.pone.0115202.g001

Fig. 2. TEM images of magnetic nanoparticles. MNPs were coated with (a) OH, and (b) NH2 groups.Samples were dispersed in ethanol using an ultrasound bath and then placed onto a Formvar-covered coppergrid and evaporated in air at room temperature. Bars represent 20 nm in both pictures.




characteristics, and corresponds to 75 K, [37]) and the other one at close to room

temperature (Fig. 3). Both types of nanoparticles (MNPOH and MNPNH2)

showed hysteresis behavior below the blocking temperature, although this

characteristic disappeared at 300 K. At this temperature, magnetic remanence

(Mr) and coercivity (Hc) values fell to near zero, which is typical of

superparamagnetic nanoparticles. The saturation magnetization (Ms) was

Table 1. Main characteristics of MNPs coated with OH and NH2 groups.

MNPOH MNPNH2

Diameter (nm) 10.40.9 10.30.9

Surface area (m2/g) 112.79.3 113.89.4

NH2 group density (mmol/g) 0.240.01 42.960.02

Ms (emu/g) 86.0 82.5

Hc (Oe) 0.3 0.1

Mr (emu/g) 3.4 1.0

doi:10.1371/journal.pone.0115202.t001

Fig. 3. Magnetization analysis of magnetic nanoparticles at different temperatures. Magnetization analysis of MNPOH (black lines) and MNPNH2(grey lines) were performed at 300 K (solid lines) and 2 K (dotted lines). Inlet shows a detail of magnetization for values of magnetic field near to 0 Oe.




determined to be 86.0 and 82.5 emu/g for naked and NH2functionalized

nanoparticles, respectively, which is lower than that reported for bulk magnetite

(89 emu/g, [38]). This decrease in Ms was also observed by other authors [26, 39]

and could be related to the difference between macroscopic and nanoscopic

magnetite [25, 27, 35, 39]. On the other hand, the binding of APTS to the

nanoparticles caused a slight decrease of 4% in the Ms of the magnetic support,

which has been previously observed when organic molecules were immobilized on

MNPs [40, 41]. This fact could be explained because the binding of APTS on the

nanoparticle surface might quench the magnetic moment [42, 43]. Fitting

magnetic result to the Langevins function, a magnetic core average diameter of

7.8 nm, with 34 nm dispersion, was calculated. This value is consistent with the

size determined from XRD data as aforementioned.

Preparation of mCLEAsSynthesis of mCLEAs involves a sequence of steps starting with enzyme

insolubilization followed by cross-linking the resulting protein aggregates with

themselves and the surfaces of NH2-functionalized magnetic nanoparticles.

Optimization aspects concerning the selection of precipitation conditions, cross-

linking agent, time or enzyme concentration were considered.

Effect of precipitating agent

Commonly used lipase precipitating agents were selected to insolubilize CALB,

i.e., ethanol, 2-propanol, acetone and ammonium sulphate (125 mmol/mg

protein). After enzyme precipitation, mCLEAs were prepared at room

temperature by cross-linking (for 24 h) 0.2 mg CALB/mg MNPNH2 in the

presence of 200 mM glutaraldehyde. A control using soluble enzyme was carried

out in parallel, with initial protein concentration of 0.133 mg/ml and esterase

activity of 2.532 U/ml. The end of the immobilization process was verified by

analyzing the protein concentration in the liquid phase of the immobilization

medium, and the esterase activity in both the liquid and formed mCLEAs. The

selected precipitating agents allowed the formation of mCLEAs with 100% protein

and esterase activity retention, with the exception of ethanol, which caused a drop

in both the retained protein and esterase activity to 46.3% and 46.8%,

respectively. In addition to ethanol, also 2-propanol and acetone strongly affected

the esterase activity of the biocatalyst, resulting in values of 0.826, 0.655 and

0.383 U/mg MNPNH2. However, the mCLEAs obtained with ammonium

sulphate showed an activity of 1.823 U/mg MNPNH2. When comparing these

values with the theoretical immobilized activity (3.8 U/mg MNPNH2),

immobilization efficiencies of 21.7, 17.2, 10.1 and 48.0% were obtained when

using ethanol, 2-propanol, acetone and ammonium sulphate, respectively. These

results are in agreement with those reported by other authors [10, 11, 44], who

proposed that ammonium sulphate was the best option because it usually

preserves the enzymatic activity of the biocatalyst. Therefore, ammonium sulphate

was selected in this work as the precipitating agent to prepare mCLEAs of CALB.



The insolubilizing effect of ammonium sulphate concentration (12.5

125 mmol/mg protein) was studied at room temperature by measuring the

esterase activity retained by the resulting mCLEAs prepared using 50 mg to 1.2 mg

lipase/mg MNPNH2 and cross-linked for 5 h with 200 mM glutaraldehyde. At

low concentration of lipase a good relationship between the immobilized esterase

activity and ammonium sulphate was observed (Fig. 4). However at the highest

concentration of salt (125 mmol/mg protein), lower activity recovery was

measured at lipase concentrations higher than 0.4 mg protein/mg MNPNH2.

This effect is probably due to excessive cross-linking and/or size of aggregates

which would lead to irreversible enzyme denaturation and/or the appearance of

diffusional problems.

It should be noted that the theoretical immobilized activity increases with the

increase of enzyme concentration in the preparation. Maximum esterase activities

of 0.95, 1.91, 3.82, 7.67, 15.27 and 22.91 U/mg MNPNH2 correspond to protein

concentrations of 0.05, 0.1, 0.2, 0.4, 0.8 and 1.2 mg protein/mg MNPNH2,

respectively. Maximum immobilization efficiencies of around 60% were obtained

when 0.1 mg protein/mg MNPNH2 were used. Further increase in enzyme

concentrations was not reflected in an increase of recovery activity. This is a usual

behavior of immobilized enzymes which is related to diffusional limitations

occurring when diffusional rate of substrates and products is lower than reaction

rate, thus decreasing the observed rate of the reaction.

Conclusively, 0.1 mg protein/mg MNPNH2 and 100 mmol of ammonium

sulphate/mg protein were selected in order to prepare mCLEAs of CALB.

Fig. 4. Combined effect of CALB and insolubilizing agent concentrations on the esterase activity ofmCLEAs. Different concentrations of lipase were cross-linked for 5 h with 200 mM glutaraldehyde in thepresence of the indicated concentrations of ammonium sulphate.




Effect of detergents

It is well known that the presence of a detergent at an appropriate concentration

can often hyperactivate the enzyme, especially when using lipases [4547].

Furthermore, detergents may prevent the enzyme immobilization through its

active center and promote the enzyme orientation in the support so enhancing its

activity and/or stability.

The esterase activity of soluble CALB was measured in the presence of Triton X-

100, Tween 20, Tween 80 and SDS, at different concentrations below and above

their critical micelle concentration (cmc) which were 0.6, 0.06, 0.012 and 8 mM

(0.388, 0.074, 0.016 and 2.307 kg/m3), respectively. The activity of soluble CALB

increased in the presence of Triton X-100 at concentrations above its cmc (S1A

Fig.). However, the presence of Triton X-100 at such concentrations during the

preparation of mCLEAs had a negative effect in oil transesterification (S1b Fig.).

Fernandez-Lorente et al. [45] also immobilized CALB in the presence of Triton X-

100 on an aminated support, with a final detergent concentration of 1% (v/v) (i.e.,

17 mM, 11 kg/m3), observing an increase in hydrolytic activity, but this was not

tested in organic media [45, 46]. The detergent could enhance enzymatic activity

in aqueous medium reactions, but it could lose the hyperactivation capability in

hydrophobic media, due to the formation of inverse micelles.

Both Tween 20 and Tween 80 had no effect on CALB esterase activity nor on

mCLEAs transesterification activity. The presence of SDS decreased the activity of

poorly concentrated mCLEAs, whereas apparently caused a strong hyperactivation

of highly concentrated samples. Such hyperactivation is similar to that described

by Lopez-Serrano et al. [48] and Gupta et al. [44], who prepared CLEAs in the

presence of SDS and observed that the activity increased 23 fold. Actually, the

transesterification activity of the biocatalyst prepared with high concentration of

protein in absence of SDS should be 4-fold higher than the one with lower

concentration. The presence of SDS when preparing mCLEAs with highly

concentrated aggregates could protect the hydrophobic areas close to active

centers, resulting in biocatalysts with lower steric hindrance and thus, higher

values of specific activity.

The use of detergents during the formation of mCLEAs would only be

recommended for the immobilization of high protein concentrations. However,

detergents were not used for further experiments in this work as concentrations of

protein above 0.1 mg/mg MNPNH2 seem to result in low immobilized enzyme

retentions.

Effect of cross-linker concentration

The cross-linking effect of glutaraldehyde was investigated by assessing the

esterase activity of mCLEAs prepared by incubating for 5 h the insolubilized lipase

(0.1 mg CALB/mg MNPNH2 with 100 mmol ammonium sulphate/mg protein)

in the presence of glutaraldehyde at concentrations from 0 to 500 mM (Table 2).

The immobilized esterase activity increased linearly when glutaraldehyde

concentration was varied from 0 to 200 mM, the maximum value (1.24 U/mg

MNPNH2) representing 65% of offered activity (1.91 U/mg MNPNH2). Above



that concentration, the immobilized activity remained unchanged. At higher

glutaraldehyde concentration, the cross-linking of MNPNH2 could be favored,

reducing the available surface area for the immobilization of aggregates. On the

other hand, the immobilization time of 5 h could be insufficient for the formation

of covalent bonds, and a more prolonged incubation at these conditions should be

used in order to increase the activity retention.

Effect of protein concentration and immobilization time

At first approach, mCLEAs of CALB (0.1 mg protein/mg MNPNH2, 100 mmol

sulphate/mg protein, 200 mM glutaraldehyde, no detergent) were prepared by

varying the time (0, 0.5, 1, 2, 3, 4, 5, 6 and 24 h) used to cross-link the enzyme

aggregates onto the surface of MNPNH2. Addition of the cross-linker agent

resulted in an immediate activity retention of 42%. This value rose from 65% to

almost 100% when the contact time was enlarged from 5 h to 24 h (Table 3). This

increase indicates that the immobilization process requires large periods of time to

completely establish covalent linkages between enzyme and support. With this in

mind, the observed apparent low value of retained activity when high loads of

protein were immobilized for 5 h (Table 3) could be due to low retention of

protein during this insufficient period of time. When an immobilization time of

24 h was used, the complete protein immobilization was confirmed by analysis of

unbound protein at the different steps of the process (S2 Fig.). Even so, high

protein loads resulted in low apparent retained activities. Data suggest that 0.1 mg

protein/mg MNPNH2 is the maximum concentration that ensures the absence of

steric hindrance observed in esterase activity measurement.

Lipases have been immobilized in a great variety of supports in several works

[10, 49], obtaining immobilization efficiencies below 80%. Xie and Ma [18, 19]

immobilized lipase on amino-functionalized magnetic nanoparticles and studied

the optimization of the protein concentration to obtain maximum immobiliza-

tion efficiencies. As a result, they obtained 70% efficiency at concentrations in the

Table 2. Effect of cross-linker concentration on the preparation of mCLEAs of CALB.

Glutaraldehyde (mM) Recovered activity (U/mg MNPNH2) Apparent retained activity (%)

0 0.021 1.1

5 0.019 1.0

10 0.031 1.6

25 0.079 4.1

75 0.391 20.5

125 0.893 46.8

200 1.238 64.8

250 1.188 62.2

300 1.180 61.8

350 1.285 67.3

400 1.315 68.8

500 1.142 59.8




range of 0.025 to 0.05 mg protein/mg MNPNH2. Our improved immobilization

method allowed the load of a higher concentration of protein per mg of support

with total activity retention, which leads to enzyme and support savings, and

consequently economical benefits in production processes.

Morphology of mCLEAs

The size and shape of mCLEAs of CALB were examined by TEM after staining the

samples with 1% (w/v) phosphotungstic acid. Discrete arrangements of about

100 nm675 nm (Fig. 5a) were routinely detected although larger aggregatescould be also observed (Fig. 5b).

Biodiesel production by mCLEAsThe ability of the magnetic CLEAs to catalyze the production of biodiesel from

vegetable oils in a solvent-free system was tested. The transesterification reaction

was performed using 200 mg olive oil and 2-propanol in a molar ratio of 6:1

(alcohol:oil) in the presence of different concentrations of mCLEAs (prepared

using 0.1 mg protein/mg MNPNH2, 100 mmol sulphate/mg protein, 200 mM

glutaraldehyde, no detergent). After incubating the reaction mixtures containing

mCLEAs (at 0.5, 1, 2 and 5% w/w of oil) for 24 h at 30 C, conversions of 64.0,

76.1, 81.5 and 82.2%, respectively, were obtained. These data indicate that a very

low concentration of the biocatalyst (1% w/w of oil) is suitable to catalyze the

transesterification of olive oil.

The time-course of the reaction was assessed during 72 h for both control olive

oil and waste frying oil. Samples were withdrawn at time intervals and analyzed by

either HPLC (olive oil) or TLC (waste frying oil). HPLC data revealed the

appearance of diglycerides, monoglycerides and free fatty acids (Fig. 6) as

intermediates in the synthesis of biodiesel (fatty acid propyl esters, FAPEs). These

intermediates could be also observed in samples analyzed by TLC and stained with

Table 3. Effect of protein concentration and immobilization time on the preparation of mCLEAs of CALB.

Immobilization time (h)Offered protein (mg/mgMNPNH2)

Offered activity (U/mgMNPNH2)

Recovered activity (U/mgMNPNH2)

Apparent retained activity(%)

5 0.05 0.95 0.63 66.0

0.1 1.91 1.24 65.0

0.2 3.82 1.55 40.5

0.4 7.67 1.63 21.3

0.8 15.27 1.79 11.7

1.2 22.91 1.57 6.9

24 0.05 0.95 0.93 97.4

0.1 1.91 1.90 99.5

0.2 3.82 1.82 47.8

0.4 7.67 2.08 27.2




Coomassie Blue (Fig. 7a), indicating that this versatile semi-quantitative method

can be useful to determine the reaction progress with time by following changes in

substrate and products concentrations. It is worth mentioning that TLC analysis

followed by plate revealing with Coomassie Blue permits to analyze in parallel at

least 10 samples in less than 1 h. In addition it allows the use of crude and/or

recycled vegetable oils which could originate troubles when analyzed by HPLC.

The efficiency of low concentrations of mCLEAs to catalyze the conversion into

biodiesel of other non-edible vegetable triglycerides was tested using jatropha,

cameline and crude (unrefined) soybean oils. Samples of these crude oils were

used without any pretreatment. The transesterification reaction with 2-propanol

was carried out using 1% of biocatalyst (w/w oil) at 30 C for 72 h, as described

previously for olive oils, and the resulting FAPEs were analyzed by TLC. As shown

in Fig. 7b, similar bioconversions were observed in all cases after 24 h of

transesterification reaction.

The immobilization of lipase to different carriers for biodiesel production has

been studied previously with high conversion results (64100%), although

elevated concentrations of biocatalyst (515% w/w of oil) were used in most of the

cases [4952]. Even when lipase was immobilized on NH2-functionalized

Fig. 5. TEM images of mCLEAs. Samples were negatively stained with phosphotungstic acid and dispersedin ethanol as in Fig. 2. (a) and (b) represent different images of the same sample. Bars represent 50 nm inboth pictures.




Fig. 6. Time-course of biodiesel (FAPEs) conversion catalyzed by mCLEAs of CALB. The reactionmixture containing 200 mg olive oil, 2-propanol (6:1 alcohol:oil molar ratio) and 1% (w/w of oil) mCLEAs wasincubated for 72 h at 30C with rotational mixing. Samples (10 ml) were withdrawn at the indicated times andanalyzed by HPLC. Triglycerides (N), FAPEs (&), diglycerides (n) and monoglycerides with free fatty acids(e).


Fig. 7. Analysis by TLC of biodiesel (FAPEs) conversion using mCLEAs of CALB. The reaction mixture and conditions were the same as in Fig. 6,except that (a) waste frying olive oil and (b) different vegetable oils were used as substrate. Olive oil was included as a control substrate. Triglycerides (TGs),diglycerides (DGs), monoglycerides (MGs) and free-fatty acids (FFAs).




magnetic nanoparticles, requirements from 17.3 to 40% (w/w of oil) of biocatalyst

were reported [1820]. Only a few number of researchers described the use of

lower concentrations of enzyme to catalyze the transesterification reaction, as

Mendes et al. [53] who used 0.2% (w/w of oil) of enzyme, although they worked

at 45 C with high alcohol:oil molar ratios. It is well known that temperature not

only increases kinetics but also can affect stability of the enzyme.

The stability and reusability of mCLEAs of CALB was investigated by reusing

the biocatalyst for several consecutive reaction cycles at temperatures from 30 to

60 C. After each cycle (24 h) the biocatalyst was recovered from the reaction

mixture using a magnetic field. Two different catalysts, prepared using 0.05 and

0.1 mg CALB/mg MNPNH2, were tested. The biocatalyst with lower concen-

tration of lipase not only showed a 4-fold lower transesterification activity but it

was also considerably less stable than its counterpart containing 0.1 mg protein/

mg MNPNH2 (Fig. 8). Using the latter, the 24 h-conversion was preserved for at

least 10 consecutive reaction cycles without observing any significant loss of

catalytic capacity. Although stability decreased with the increase in temperature,

the highly-loaded biocatalyst maintained more than 60% of the activity after 10

cycles at 60 C.

Wang et al. [20] observed that the retained activity of their lipase catalyst

decreased by more than 70% after 10 consecutive cycles of reaction at 40 C; Xie

and Ma [18, 19] observed a decrease of 50% activity after the fifth cycle of catalysis

using Thermomyces lanuginosus lipase immobilized on MNPs for the production

of biodiesel at high temperatures (4550 C). Our data reveal that the combination

of cross-linked enzyme aggregates with MNPs results in a robust biocatalyst which

is able to catalyze the synthesis of biodiesel at low enzyme concentration, and is

more stable than other lipase preparations reported so far. The effectiveness of this

biocatalyst for the synthesis of biosurfactants was also proved recently in a

previous work from our group [54].

Novel enzyme engineering techniques are being applied for the improvement of

catalytic efficiencies of CALB. Thus, Agarwal and coworkers [55] introduced a

pothoactivatable molecule on the surface of the enzyme, which allowed the

modulation of the enzyme conformation by light stimulation, obtaining an

increase in 24% of CALB catalytic activity. A combination of protein engineering

with mCLEAs formation could result in an easily recoverable and reusable enzyme

with improved enzymatic activity and high stability, which could considerably

increase the robustness and applicability of the biocatalyst.

Conclusions

In this work we report an efficient, facile and economical method to prepare

mCLEAs of Candida rugosa lipase B by cross-linking with glutaraldehyde CALB

aggregates onto the surface of MNPs functionalized with -NH2 groups. The

resulting magnetic biocatalyst shows an enhanced stability and maintains the

superparamagnetic behavior typical of MNPs, which allows for easy separation



and reusability in consecutive catalytic cycles without apparent loss of activity.

The utility of this robust biocatalyst for the efficiently production of biodiesel has

been demonstrated although it could be also used to obtain other highly valuable

compounds, such as biosurfactants, flavoring and aromatic compounds or

bioactive compounds.

Fig. 8. Reusability of mCLEAs of CALB at different temperatures. Biocatalyst was used in consecutivereaction cycles of 24 h for biodiesel (FAPEs) conversion from olive oil at: (a) 30C, (b) 40C, (c) 50C and (d)60C. The reaction mixture and conditions were the same as in Fig. 6. Relative conversion is referred to thefinal conversion after the first cycle and was calculated using samples analyzed by HPLC. Two types ofmCLEAs were tested which were prepared using 0.05 (q) and 0.1 mg protein/mg MNP-NH2 (N).doi:10.1371/journal.pone.0115202.g008



Supporting Information

S1 Fig. Effect of detergents on enzymatic activity. (a) Relative esterase activity of

soluble CALB; (b) Oil transesterification activity of mCLEAs: (N) 0.1 mg CALB/mg MNP2NH2; (q) 0.4 mg CALB/mg MNP2NH2.

doi:10.1371/journal.pone.0115202.s001 (TIF)

S2 Fig. Analysis of unbound protein after the different immobilization steps: (a)

MNP-CALB (50 mg CALB/mg MNP-NH2 in absence of precipitant agent); (b)

mCLEAs (100 mg CALB/mg MNP-NH2). Cont: control of offered protein (CALB,

molecular mass 533.5 kDa); UR: unretained protein after 2 h of cross-linking; 1:

first wash with PBS; 2: second wash with PBS; 3: third wash with PBS; BH4:

unretained protein after NaBH4 reduction; NaCl: unretained protein after

washing with 2 M NaCl; TX: unretained protein after washing with 1% (v/v)

Triton X-100; B: liquid phase after incubating the MNP-CALB complex for 5 min

at 100 C.

doi:10.1371/journal.pone.0115202.s002 (TIF)

S1 Video. Synthesis of magnetic nanoparticles.

doi:10.1371/journal.pone.0115202.s003 (MPG)

Acknowledgments

We are grateful to Mr Ramiro Martnez (Novozymes A/S, Spain) for providing us

with CALB samples.

Author ContributionsConceived and designed the experiments: ACI CL JLS MJL. Performed the

experiments: ACI EAP CL. Analyzed the data: ACI EAP CL JLS MJL. Contributed

reagents/materials/analysis tools: ACI EAP CL JLS MJL. Wrote the paper: ACI CL

JLS MJL.

References

1. Bajaj A, Lohan P, Jha PN, Mehrotra R (2010) Biodiesel production through lipase catalyzedtransesterification: An overview. J Mol Catal B Enzym 62: 914.

2. Leung DYC, Wu X, Leung MKH (2010) A review on biodiesel production using catalyzedtransesterification. Appl Energy 87: 10831095.

3. Bisen PS, Sanodiya BS, Thakur GS, Baghel RK, Prasad GB (2010) Biodiesel production with specialemphasis on lipase-catalyzed transesterification. Biotechnol Lett 32: 10191030.

4. Ren Y, Rivera JG, He L, Kulkarni H, Lee DK, et al. (2011) Facile, high efficiency immobilization oflipase enzyme on magnetic iron oxide nanoparticles via a biomimetic coating. BMC Biotechnol 11: 63.

5. Idris A, Bukhari A (2012) Immobilized Candida antarctica lipase B: Hydration, stripping off andapplication in ring opening polyester synthesis. Biotechnol Adv 30: 550563.

6. Netto CGCM, Andrade LH, Toma HE (2009) Enantioselective transesterification catalysis by Candidaantarctica lipase immobilized on superparamagnetic nanoparticles. Tetrahedron: Asymmetry 20: 22992304.



7. Ngo TPN, Li A, Tiew KW, Li Z (2013) Efficient transformation of grease to biodiesel using highly activeand easily recyclable magnetic nanobiocatalyst aggregates. Bioresour Technol 145: 233239.

8. Netto CGCM, Toma HE, Andrade LH (2013) Superparamagnetic nanoparticles as versatile carriers andsupporting materials for enzymes. J Mol Catal B: Enzym 8586: 7192.

9. Verma ML, Barrow CJ, Puri M (2013) Nanobiotechnology as a novel paradigm for enzymeimmobilisation and stabilisation with potential applications in biodiesel production. Appl MicrobiolBiotechnol 97: 2339.

10. Prabhavathi Devi BLA, Guo Z, Xu X (2009) Characterization of cross-linked lipase aggregates. J AmOil Chem Soc 86: 637642.

11. Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom APG, et al. (2004) Preparation,optimization, and structures of cross-linked enzyme aggregates (CLEAs). Biotechnol Bioeng 87: 754762.

12. Contesini FJ, Figueira JdA, Kawaguti HY, Fernandes PCdB, Carvalho PdO, et al. (2013) Potentialapplications of carbohydrases immobilization in the food industry. Int J Mol Sci 14: 13351369.

13. Sheldon RA (2007) Cross-linked enzyme aggregates (CLEAs): stable and recyclable biocatalysts.Biochem Soc Trans 35: 15831587.

14. Sheldon RA (2011) Characteristic features and biotechnological applications of cross-linked enzymeaggregates (CLEAs). Appl Microbiol Biotechnol 92: 467477.

15. Sheldon RA, van Pelt S (2013) Enzyme immobilization in biocatalysis: why, what and how. Chem SocRev 42: 62236235.

16. Talekar S, Ghodake V, Ghotage T, Rathod P, Deshmukh P, et al. (2012) Novel magnetic cross-linkedenzyme aggregates (magnetic CLEAs) of alpha amylase. Bioresour Technol 123: 542547.

17. Talekar S, Joshi A, Joshi G, Kamat P, Haripurkar R, et al. (2013) Parameters in preparation andcharacterization of cross-linked enzyme aggregates (CLEAs). RSC Adv 3: 1248512511.

18. Xie W, Ma N (2009) Immobilized lipase on Fe3O4 nanoparticles as biocatalyst for biodiesel production.Energy Fuels 23: 13471353.

19. Xie W, Ma N (2010) Enzymatic transesterification of soybean oil by using immobilized lipase onmagnetic nano-particles. Biomass Bioenergy 34: 890896.

20. Wang X, Dou P, Zhao P, Zhao C (2009) Immobilization of lipases onto magnetic Fe3O4 nanoparticles forapplication in biodiesel production. ChemSusChem 2: 947950.

21. Han JY, Kim HK (2011) Transesterification using the cross-linked enzyme aggregate of Photobacteriumlipolyticum lipase M37. J Microbiol Biotechnol 21: 11591165.

22. Lai J-Q, Hu ZL, Sheldon RA, Yang Z (2012) Catalytic performance of cross-linked enzyme aggregatesof Penicillium expansum lipase and their use as catalyst for biodiesel production. Process Biochem 47:20582063.

23. Cruz-Izquierdo A, Pico EA, Anton-Helas Z, Boeriu CG, Llama MJ, et al. (2012) Lipase immobilizationto magnetic nanoparticles: methods, properties and applications for biobased products. New Biotechnol29S: S100S101.

24. Tudorache M, Nae A, Coman S, Parvulescu V (2013) Strategy of cross-linked enzyme aggregatesonto magnetic particles adapted to the green design of biocatalytic synthesis of glycerol carbonate. RSCAdv 3: 40524058.

25. Morales MdP, Veintemillas-Verdaguer S, Montero MI, Serna CJ, Roig A, et al. (1999) Surface andinternal spin canting in c-Fe2O3 nanoparticles. Chem Mater 11: 30583064.

26. Shen X-C, Fang X-Z, Zhou Y-H, Liang H (2004) Synthesis and characterization of 3-aminopropyltriethoxysilane-modified superparamagnetic magnetite nanoparticles. Chem Lett 33:14681469.

27. Liu ZL, Liu YJ, Yao KL, Ding ZH, Tao J, et al. (2002) Synthesis and magnetic properties of Fe3O4nanoparticles. J Mater Synth Process 10: 8387.

28. del Campo A, Sena T, Lellouche J-P, Bruce IJ (2005) Multifunctional magnetite and silicamagnetitenanoparticles: synthesis, surface activation and applications in life sciences. J Magn Magn Mater 293:3340.



29. Mateo C, Fernandez-Lorente G, Abian O, Fernandez-Lafuente R, Guisan JM (2000) Multifunctionalepoxy supports: a new tool to improve the covalent immobilization of proteins. The promotion of physicaladsorptions of proteins on the supports before their covalent linkage. Biomacromolecules 1: 739745.

30. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophageT4. Nature 277: 680685.

31. Gao L, Xu J-H, Li X-J, Liu Z-Z (2004) Optimization of Serratia marcescens lipase production forenantioselective hydrolysis of 3-phenylglycidic acid ester. J Ind Microbiol Biotechnol 31: 525530.

32. Holcapek M, Jandera P, Fischer J, Prokes B (1999) Analytical monitoring of the production of biodieselby high-performance liquid chromatography with various detection methods. J Chromatogr A 858: 1331.

33. Samakawa T, Kaieda M, Matsumoto T, Ban K, Kondo A, et al. (2000) Pretreatment of immobilizedCandida antarctica lipase for biodiesel fuel production from plant oil. J Biosci Bioeng 90: 180183.

34. Nakamura K, Handa S (1984) Coomassie brilliant blue staining of lipids on thin-layer plates. AnalBiochem 142: 406410.

35. Harris LA, Goff JD, Carmichael AY, Riffle JS, Harburn JJ, et al. (2003) Magnetite nanoparticledispersions stabilized with triblock copolymers. Chem Mater 15: 13671377.

36. Sahoo Y, Goodarzi A, Swihart MT, Ohulchanskyy TY, Kaur N, et al. (2005) Aqueous ferrofluid ofmagnetite nanoparticles: Fluorescence labeling and magnetophoretic control. J Phys Chem 109: 38793885.

37. Lee J, Na HB, Kim BC, Lee JH, Lee B, et al. (2009) Magnetically-separable and highly-stable enzymesystem based on crosslinked enzyme aggregates shipped in magnetite-coated mesoporous silica.J Mater Chem 19: 78647870.

38. Ramrez LP, Landfester K (2003) Magnetic polystyrene nanoparticles with a high magnetite contentobtained by miniemulsion processes. Macromol Chem Phys 204: 2231.

39. Maity D, Agrawal DC (2007) Synthesis of iron oxide nanoparticles under oxidizing environment andtheir stabilization in aqueous and non-aqueous media. J Magn Magn Mater 308: 4655.

40. Bayramoglu G, Kiralp S, Yilmaz M, Toppare L, Arica MY (2008) Covalent immobilization ofchloroperoxidase onto magnetic beads: Catalytic properties and stability. Biochem Eng J 38: 180188.

41. Yong Y, Bai YX, Li YF, Lin L, Cui YJ, et al. (2008) Preparation and application of polymer-graftedmagnetic nanoparticles for lipase immobilization. J Magn Magn Mater 320: 23502355.

42. Huang SH, Liao MH, Chen DH (2003) Direct binding and characterization of lipase onto magneticnanoparticles. Biotechnol Progr 19: 10951100.

43. Sohn OJ, Kim CK, Rhee JI (2008) Immobilization of glucose oxidase and lactate dehydrogenase ontomagnetic nanoparticles for bioprocess monitoring system. Biotechnol Bioprocess Eng 13: 716723.

44. Gupta P, Dutt K, Misra S, Raghuwanshi S, Saxena RK (2009) Characterization of cross-linkedimmobilized lipase from thermophilic mould Thermomyces lanuginosus using glutaraldehyde. BioresourTechnol 100: 40744076.

45. Fernandez-Lorente G, Palomo JM, Mateo C, Munilla R, Ortiz C, et al. (2006) Glutaraldehyde cross-linking of lipases adsorbed on aminated supports in the presence of detergents leads to improvedperformance. Biomacromolecules 7: 26102615.

46. Fernandez-Lorente G, Palomo JM, Cabrera Z, Fernandez-Lafuente R, Guisan JM (2007) Improvedcatalytic properties of immobilized lipases by the presence of very low concentrations of detergents inthe reaction medium. Biotechnol Bioeng 97: 242250.

47. Liu Y-Y, Xu J-H, Hu Y (2000) Enhancing effect of Tween-80 on lipase performance in enantioselectivehydrolysis of ketoprofen ester. J Mol Catal B Enzym 10: 523529.

48. Lopez-Serrano P, Cao L, van Rantwijk F, Sheldon RA (2002) Cross-linked enzyme aggregates withenhanced activity: application to lipases. Biotechnol Lett 24: 13791383.

49. Dizge N, Keskinler B (2008) Enzymatic production of biodiesel from canola oil using immobilized lipase.Biomass Bioenergy 32: 12741278.



50. Azocar L, Ciudad G, Heipieper HJ, Munoz R, Navia R (2010) Improving fatty acid methyl esterproduction yield in a lipase-catalyzed process using waste frying oils as feedstock. J Biosci Bioeng 109:609614.

51. Charpe TW, Rathod VK (2011) Biodiesel production using waste frying oil. Waste Manage 31: 8590.

52. Iso M, Chen B, Eguchi M, Kudo T, Shrestha S (2001) Production of biodiesel fuel from triglyceridesand alcohol using immobilized lipase. J Mol Catal B Enzym 16: 5358.

53. Mendes AA, Giordano RC, Giordano RdLC, Castro HFd (2011) Immobilization and stabilization ofmicrobial lipases by multipoint covalent attachment on aldehyde-resin affinity: Application of thebiocatalysts in biodiesel synthesis. J Mol Catal B: Enzym 68: 109115.

54. Lopez C, Cruz-Izquierdo A, Pico EA, Garca-Barcena T, Villarroel N, et al. (2014) Magneticbiocatalysts and their uses to obtain biodiesel and biosurfactants. Front Chem 2: 72.

55. Agarwal PK, Schultz C, Kalivretenos A, Ghosh B, Broedel SE (2012) Engineering a hyper-catalyticenzyme by photoactivated conformation modulation. J Phys Chem Lett 3: 11421146.



Section_1Section_2Section_3Section_4Section_5Equation equ1Equation equ2Section_6Section_7Section_7aSection_7bSection_7cSection_8Section_8aSection_9Section_10Section_11Figure 1Figure 2TABLE_1Figure 3Section_12Section_13Section_14Figure 4Section_15Section_16TABLE_2Section_17Section_18TABLE_3Figure 5Figure 6Figure 7Section_19Section_21Section_22Figure 8Section_23Section_24Section_25Section_26Section_27Section_28Reference 1Reference 2Reference 3Reference 4Reference 5Reference 6Reference 7Reference 8Reference 9Reference 10Reference 11Reference 12Reference 13Reference 14Reference 15Reference 16Reference 17Reference 18Reference 19Reference 20Reference 21Reference 22Reference 23Reference 24Reference 25Reference 26Reference 27Reference 28Reference 29Reference 30Reference 31Reference 32Reference 33Reference 34Reference 35Reference 36Reference 37Reference 38Reference 39Reference 40Reference 41Reference 42Reference 43Reference 44Reference 45Reference 46Reference 47Reference 48Reference 49Reference 50Reference 51Reference 52Reference 53Reference 54Reference 55

magnetic cross-linked enzyme aggregates (cleas) of candida antarctica lipase. an efficient and...

Documents

magnetic biocatalyst

magnetic character

department of chemistry

university of bath

h of reaction

nonedible vegetable

ismore stable

free enzyme